MIRANDA Maria Victoria
Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method.
A. TARGOVNIK; L. ROMERO; F. WOLMAN; O. CASCONE; M. V. MIRANDA
PROCESS BIOCHEMISTRY - (Print)
ELSEVIER SCI LTD
Año: 2010 vol. 45 p. 835 - 835
Horseradish peroxidase is useful in many biotechnological fields, including diagnostics, biocatalysis and biosensors. Horseradish peroxidase isozyme C (HRPC) extracellular expression in Spodoptera frugiperda Sf9 cell culture and in intact larvae is shown. At day 6 post infection, active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 x 106 cells or 3.0 ± 0.1 mg l-1 with a multiplicity of infection of 1 in the presence of 7.2 µM hemin. Similar yields were obtained in monolayer cultures. In larvae, HRPC expression level was 137 ± 17 mg HRPC Kg-1 larvae at day 6 post infection, HRPC produced by a single larva was around 41 μg. The whole larvae extract was directly subjected to ion exchange chromatography and HRPC was purified in only one step with a yield of 75% and a purification factor of 117. Recombinant HRPC molecular weight was 44,016 Da and its glycosylation pattern agrees with that expected for invertebrates. The Km and Vmax were 12.1 ± 1.7 mM and 2673 ± 113 U mg-1 respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study based on S. frugiperda larvae is an inexpensive and simple way to obtain high levels of active HRPC for research and other biotechnological applications.